The overall goal of this research is to elucidate the signaling pathways responsible for protective adaptation. Brief periods of intermittent exposure to stress (known as preconditioning (PC)) have been shown to provide protection against injury during a subsequent more severe stress. We have investigated several signaling pathways which might be involved in the protection afforded by PC. We reported that 12-HETE, the stable end-product of lipoxygenase metabolism, is made during the preconditioning protocol, and that inhibitors of the 12-lipoxygenase also blocks preconditioning. We also find that an alteration in redox state appears to be involved in preconditioning. Preconditioning results in a slight, but significant decrease in the GSH/GSSG, and addition of the antioxidant and glutathione precursor, N-acetyl-cysteine during the preconditioning protocol is found to block the protective effects of preconditioning. Furthermore, diamide and BSO given for 5 minutes prior to sustained ischemia mimic preconditioning. We also find that the endogenous PKC activator, DOG, can mimic preconditioning, and that the PKC inhibitor, chelerythrine blocks the protection afford by preconditioning when it is added during the preconditioning protocol. We are currently investigating whether activation of PKC occurs upstream or downstream of changes in redox or production of lipoxygenase metabolites. Preliminary data suggest that preconditioning activates PKC which then leads to an increase in 12-lipoxygenase metabolites. We find that PKC activation increases 12-HETE concomitant with protection. To directly investigate the involvement of the 12-lipoxygenase pathway in protective adaptation we are beginning a collaboration with Dr. Colin Funk at the University of Pennsylvania to study 12-lipoxygenase knock out mice. This new initiative first required us to adapt our perfusion and NMR methodology to mouse hearts. We can now successfully perfuse mouse hearts and obtain 31P NMR measurements with 5 minute time resolution and we can simultaneously measure left ventricular developed pressure (LVDP) via a fluid filled balloon in the left ventricle which is connected to a pressure transducer. We obtain LVDP values of 80-100 cm/water. We have also investigated the regulation of sarcoplasmic reticulum (SR) ionized calcium concentration and its role in injury and protection. We recently described the first direct measurement of sarcoplasmic reticulum (SR) ionized calcium concentration. We used an NMR sensitive, high Kd indicator which loads into SR in perfused rabbit hearts. We find that basal SR ionized calcium is 1 mM, a value consistent with the binding constant for calsequestrin, the major SR calcium binding protein. We are currently investigating mechanisms responsible for regulating SR calcium such as FK506 binding protein.